Laparoscopic Oocyte Retrieval and Cryopreservation during Vaginoplasty for Treatment of Mayer-Rokitansky-Kuster-Hauser Syndrome

Our approach allows to minimize invasive sickness in the treatment of Mayer-Rokitansky-Kuster-Hauser syndrome by combining laparoscopic vaginoplasty and oocyte retrieval for future fertility treatments. The main advantages of our approach are the requirement for a single anesthesia and the overcoming of the infeasibility of a transvaginal oocyte retrieval in younger Rokitansky patients. The procedure will be demonstrated by Diana Delprato, a senior gynecologist of our infertility unit and by Alessandra Alteri and Greta Cermisoni, two senior clinical embryologists from our assisted reproduction lab.

Start controlled ovarian stimulation on day 14 from the day of surgery. Monitor ovarian response by serial transabdominal ultrasound and concomitant measurements of estradiol and progesterone. Establish pneumoperitoneum with an open technique.

Position one 10-millimeter umbilical trocar for insertion of the laparoscope and three five-millimeter trocars in the central, right, and left lower quadrants. Under laparoscopic vision, use a 17-gauge single lumen needle connected to an aspiration pump, as commonly employed for transvaginal retrieval through the central and left trocars for puncture of the right and left ovary, respectively. Lift and hold the ovaries with laparoscopic forceps to facilitate exposure of the follicles.

When aspirating multiple follicles that are one close to another, retain the needle tip in the ovary to reduce the number of times that the ovarian cortex is transfixed and the inherent risk of bleeding. Dissect the peritoneum by lifting and incising the peritoneal strand transversely between the two rudimental uterine horns, and then, extending the incision laterally, anteriorly, and posteriorly. Expose the vaginal dimple on the perineal approach and perform an H-shaped incision and create a neo-vaginal space by sharp and blunt dissection of the vesicorectal space.

Then, pull down the dissected peritoneal margins to the edge of the vaginal vestibulum. Position interrupted sutures in polydioxanone synthetic absorbable 3-0 for the peritoneal vestibular anastomosis. Begin a purse string suture in polydioxanone synthetic absorbable 2-0 monofilament in each hemipelvis from the mobilized peritoneum above the bladder with connective transfixion of the round ligament, the tubal isthmus, the utero-ovarian ligament, and the lateral peritoneal leaf.

Then, include in the two sutures the lateral aspect of the mesorectum and the anterior aspect of the rectal serosa immediately below the rectosigmoid junction. Lastly, place a paraffin-coated gauze in the peritoneum-coated neovagina. Collect the follicular aspirates in 10-millimeter round-bottom tubes kept warm in a portable incubator and immediately transport them to the embryology laboratory with a transportation time of approximately 10 minutes.

As all the cumulus oocyte complexes are collected, place them into the four-well dish containing the fertilization medium and label the lid and the bottom with data relating to the patient's identity. Incubate them at 37 degrees Celsius in a controlled atmosphere containing 6%carbon dioxide and 5%oxygen. Ensure that a double-checking of the procedure is performed by a second member of the laboratory staff.

Perform cumulus corona cells removal within 38 hours post-ovulation trigger. Place cumulus oocyte complexes in the central well containing the enzyme to disperse the cumulus cells with a glass Pasteur pipette, gently pipetting the solution containing cumulus oocyte complexes up and down for up to 30 seconds. Move the oocytes to the second central well dish containing only HEPES buffered medium, making sure to displace a minimum amount of the enzyme.

Remove the remaining corona cells using denuding pipettes with decreasing inner diameters. Assess the stage of oocyte maturation, separating metaphase II oocytes. Transfer the metaphase II oocytes in an IVF culture 60-millimeter Petri dish containing nine drops of continuous single culture medium covered with six millimeter of mineral oil.

Incubate them at 37 degrees Celsius in a controlled atmosphere containing 6%carbon dioxide and 5%oxygen. Fill a cooling box up to the top with fresh liquid nitrogen and cover until used to minimize dispersion and evaporation. Use a stripper pipette with an inner diameter of 170 micrometers to avoid damaging oocytes during manipulation.

Gently shake each vial of equilibration solution, vitrification solution, and washing solution to mix the contents before use. Prepare the lid of a 60-milliliter Petri dish with one drop of 50 microliters of washing solution one. Place drops just before use to limit medium evaporation.

Take the oocyte dish from the incubator and transfer the metaphase II oocytes with a minimal volume of medium from the culture dish into the 50 microliters of washing solution. Dispense 50-microliter drops of washing solution two, equilibration solution one, and equilibration solution two in close proximity and transfer oocytes from drop washing solution one into drop washing solution two. Merge the drop of equilibration solution one to washing solution two and wait for two minutes for the spontaneous mixing of both the solutions.

Then, merge the drop of equilibration solution two to the previously merged drops and leave for a further two minutes. Finally, merge a new 100-microliter drop of equilibration solution three to the previously merged drops and leave for one additional minute. Place the oocytes into 100-microliter drop of equilibration solution four for 10 minutes.

Dispense two 50-microliter drops of vitrification solution and one 100-microliters of vitrification solution. Move the oocytes sequentially into the three drops of vitrification solution for 60 seconds so that the oocytes remain in each drop for approximately 20 seconds. When approximately 10 seconds are left before the end of the 60 seconds of incubation, place the cryodevice under the microscope.

Carry the oocytes at the tip of the pipette and place them on the cryodevice with the minimum amount of vitrification solution. Aspirate the excess vitrification solution, leaving the oocytes covered by a thin layer of vitrification solution. Plunge the cryodevice directly into liquid nitrogen and move it rapidly.

Keep the device in liquid nitrogen and cover it with a protective lid. The concomitant laparoscopic procedures of oocyte retrieval and vaginoplasty were combined successfully in all patients. An average of 11.4 5.4 oocytes were retrieved and 9.6 4.3 M2 oocytes were cryo-preserved.

The total average operative time was 114 17 minutes. Intraoperative blood loss was insignificant in all patients and no intraoperative adverse events were observed. On day three post-surgery, the patients began daily dilator use and were discharged on day 6.0 1.0.

A cautious aspiration of follicles within the ovary is recommended, with due attention paid to the automatic mobilization of the ovaries in order to ensure optimal access and retrieval.